The present invention relates to the removal of contaminants from PCR test sample preparations. More specifically, the present invention is directed to a process for the removal of viral fragments that could give a false positive PCR result and to the removal of interfering chemical reagents and molecules which could give a false negative result.
Blood, plasma, and biological fluid donation programs are essential first steps in the manufacture of pharmaceutical and blood products that improve the quality of life and that are used to save lives in a variety of situations. Such products are used, for example, for the treatment of immunologic disorders, for the treatment of hemophilia and in maintaining and restoring blood volume in surgical procedures and other treatment protocols. The therapeutic uses of blood, plasma, and biological fluids require the donations of these materials be as free as possible from viral contamination. Typically, a serology test sample from each individual blood, plasma, or other fluid donation is tested for various antibodies, which are elicited in response to specific viruses, such as hepatitis C (HCV) and two forms of the human immunodeficiency virus (HIV-1 and HIV-2). In addition, the serology test sample may be tested for antigens designated for specific viruses such as hepatitis B (HBV), as well as antibodies elicited in response to such viruses. If the sample is serology positive for the presence of either specific antibodies or antigens, the donation is excluded from further use.
Whereas an antigen test for certain viruses, such as hepatitis B, is thought to be closely correlated with infectivity, antibody tests are not. It has long been known that a blood plasma donor may, in fact, be infected with the virus while testing serology negative for antibodies related to that virus. For example, a window exists between the time that a donor may become infected with a virus and the appearance of antibodies, elicited in response to that virus, in the donorxe2x80x2 system. Therefore, tests directed to the detection of antibodies, may give a false indication for an infected donor if performed during the window period, i.e., the period between viral infection and the production of antibodies. Moreover, even though conventional testing for HBV includes tests for both antibodies and antigens, testing by more sensitive methods have confirmed the presence of the HBV virus in samples which were negative in the HBV antigen test. One method of testing donations, which have passed available antibody and antigen tests, in order to further ensure their freedom from incipient viral contamination, involves testing the donations by a polymerase chain reaction (PCR) method. PCR is a highly sensitive method for detecting the presence of specific DNA or RNA sequences related to a virus of interest in a biological material by amplifying the viral genome. Because the PCR test is directed to detecting the presence of an essential component of the virus itself, its presence in a donor may be found almost immediately after infection. There is, theoretically therefore, no window period during which a test may give a false indication of freedom of infectivity. A suitable description of the methodology and practical application of PCR testing is contained in U.S. Pat. Nos. 4,683,195, 4,683,202, and 5,176,995, the disclosures of which are expressly incorporated herein by reference.
Because of the exquisitely sensitive nature of the PCR test, with regard to identifying the presence of specific DNA or RNA sequences by amplifying the viral genome, PCR testing may give a false positive indication of viral activity even though no whole, i.e., infective, virus is present in the test sample. Viral genome amplification is able to occur even though the basis may merely be a number of viral fragments present in the test sample.
Commonly, blood products produced from plasma donations are subjected to a viral inactivation process before preparation of samples for PCR testing. Several well known methods are available for inactivating viruses, such as the use of solvent/detergent or heating the product to at least 60xc2x0 C. for at least 10 hours. These methods, generally, are described as being capable of reducing the concentration of viruses by a number of xe2x80x9clog units.xe2x80x9d For example, the solvent/detergent method is capable of reducing the viral contamination of the product (for example, hepatitis C) by at least 107 per ml or xe2x80x9c7 log units.xe2x80x9d Plasma products such as Factor VII Factor IX or prothrombin complex, which are derived from plasma donations, are routinely treated by heat treatment and/or the solvent/detergent method to inactivate any possible contaminating virus.
Although effective for inactivating viruses, such viral inactivation processes can result in fragmenting the whole virus, thereby rendering it non-infective while leaving the viral fragments in the product. This is particularly true when the product is heat-treated in the lyophilized state in the final container with no subsequent processing steps prior to its release. Thus, when the product is subjected to viral inactivation, particularly by heat-treatment, viral fragments remain in the product, along with the associated RNA or DNA, which may be amplified by the polymerase chain reaction. The amplification of the viral genome from non-infective viral fragments results in a false indication of viral contamination. In view of the foregoing, it can be seen that heat-treating a product for viral inactivation prior to PCR testing, but without removing the viral fragments, is not a preferred approach.
An additional source of uncertainty in PCR test results is caused by the fact that blood, plasma, serum, or blood product (such as Factor VIII or Factor IX or the like produced from blood or plasma) samples may contain significant concentrations of what might best be termed PCR inhibitors. (Both viral fragments and PCR inhibitors are referred to herein as xe2x80x9cinterfering contaminantsxe2x80x9d of the PCR process.) The most common PCR inhibitors include heme, heparin, metal ion chelators such as EDTA, and the like. Indeed, many samples are routinely collected in heparin, and it has been estimated that as much as 10% of the total sample population contains at least one form of PCR inhibitor.
Samples containing PCR inhibitors are potentially much more problematic than samples which contain significant concentrations of viral fragments. Although it is desirable to maximize the yield from a donation population, a false positive PCR viral indication would only result in one or a number of uncontaminated donations being excluded from the product stream. While very undesirable from an economic standpoint, this result does not add any substantial risk to the ultimate human consumers of the product stream. On the other hand, a false negative PCR viral indication caused by PCR inhibitors in a test sample would potentially allow a virally contaminated donation to enter the product stream.
Accordingly, in addition to removing viral fragments, a PCR sample preparation method will advantageously be able to remove PCR inhibitors as well.
A process for removing interfering contaminants, including PCR inhibitors and viral fragments, from blood protein-containing PCR assay samples is provided. The process includes the steps of introducing into a centrifuge tube an aqueous solution which comprises a blood protein and which also may include an infective virus and/or contaminants, such as viral fragments and/or PCR inhibitors in the form of soluble or suspendable chemical reagents or molecules, which interfere with the PCR assay. The aqueous blood protein solution is centrifuged to thereby separate the solution in the centrifuge tube into a supernatant which would contain potential contaminants and a solid pellet. The pellet will contain infective virus, if present, and/or insoluble protein or cellular debris or the like, depending on the nature of the sample. Substantially all of the supernatant is removed from the centrifuge tube without disturbing the pellet. An aqueous wash buffer solution is added to the centrifuge tube, thereby diluting the remaining supernatant so that potential interfering contaminants are in the diluted supernatant. The diluted supernatant is then centrifuged to provide a final supernatant and a washed pellet. A major portion of the final supernatant and any contaminants contained therein are removed from the centrifuge tube, while the residual supernatant and the washed pellet are recovered for PCR analysis.